The efficiency of many biotechnological processes is limited by the need of production organisms to balance their metabolic redox reactions. In particular, for each of the pyridine nucleotide couples (NAD/NADH and NADP/NADPH) the total rate of oxidation must be equal to the total rate of reduction: otherwise, the couple will be completely converted into one form (e.g. all in the NAD form or all in the NADH form), and reactions requiring the other form will become infinitely slow, causing the whole metabolic network of reactions be distorted in an undesirable way (i.e., no longer provide the desired product).
For example, although the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe are very efficient at converting hexoses into ethanol and have many advantages for this process (such as tolerance of high ethanol concentrations and other stresses) they are unable to ferment xylose to ethanol. Xylose is a major component of plants, and the inability to convert it to ethanol decreases the efficiency with which renewable biomass, such as agricultural wastes, can be utilized. However, these yeasts can utilize xylulose. Some yeasts (e.g. Pichia stipitis) can convert xylose to ethanol, although not very efficiently, and contain the enzymes xylose reductase (XR) and xylitol dehydrogenase (XDH). These enzymes catalyse the sequential reduction of xylose to xylitol and oxidation of xylitol to xylulose. Transformed S. cerevisiae strains have been constructed containing heterologous XR and XDH, which so possess a pathway to convert xylose into the fermentable xylulose (Kötter and Ciriacy [1993]; Tantirungkij et al. [1994]; Walfridsson et al. [1995]). Although these strains could use xylose for growth and xylitol formation, they did not produce much ethanol. All known XDH enzymes are specific for NAD, whereas all known XR enzymes are either specific for NADPH or have a preference for NADPH. It is believed (Bruinenberg et al. [1983]; Bruinenberg [1986]) that conversion of xylose to xylulose by this pathway therefore results in the cellular pool of NADPH being converted to NADP and that of NAD being converted to NADH, after which further metabolism of xylose is greatly hindered. The NADH can be reoxidised under aerobic conditions, but this demands critical control of oxygen levels to maintain fermentative metabolism and ethanol production. In contrast, bacteria that ferment xylose to ethanol efficiently contain xylose isomerase and convert xylose directly into xylulose without oxidation-reduction reactions. Attempts to create yeast that can efficiently convert xylose to ethanol have focused on finding or engineering XR or XDH enzymes with altered coenzyme specificity (Metzger and Hollenberg [1995]) or on expressing xylose isomerase gene in yeast. However, all reported attempts (see, e.g., Amore et al. [1989]; Ho et al. [1983]; Sarthy et al. [1987]; Walfridsson et al. [1996]) to construct good xylose-fermenting strains by expressing bacterial xylose isomerase genes in yeasts have failed.
As a second example, a major biotechnological process is the fermentation of hexose sugars to ethanol by yeast. The glycolytic pathway from glucose to ethanol is redox neutral, i.e. the amount of NAD reduced in the formation of a certain amount of pyruvate from glucose is exactly the same as the amount of NADH oxidised in the formation of ethanol from the same amount of pyruvate, and NADP(H) is not directly involved in the process. However, yeast growth is not a redox neutral process; the formation of 100 g dry yeast matter from glucose and ammonia is accompanied by the net production of 1.3 moles of NADH and 0.9 moles of NADP (Oura [1972]). This excess NADH is produced mainly by energy yielding catabolism, whereas the excess NADP is produced mainly by biosynthetic pathways (see Oura [1972]). Like other organisms, yeast has distinct pyridine nucleotide systems (NAD(H) and NADP(H)) that have evolved to facilitate these two aspects of metabolism. The excess NADH produced by fermenting yeast is reoxidised to NAD mainly by glycerol-3-phosphate dehydrogenase, resulting in the production of glycerol. In distillery fermentations this represents a wasteful diversion of 3–5% of the carbon source (Oura [1977]). Attempts to decrease the proportion of glycerol to ethanol produced during fermentations have met with little or no success. For example, Bjöorkqvist et al. (1997) deleted each and both of the genes encoding glycerol-3-phosphate dehydrogenase. However, yeasts lacking this enzyme were not only unable to grow under anaerobic conditions, but they also stopped making ethanol.
A third example is the biotechnological production of amino acids. Amino acids have extensive applications in the food, animal feed, medical and chemical industries. Fermentation processes have been developed to produce most amino acids occuring in proteins. The metabolic routes to amino acids first convert a carbon source such as glucose into intermediates such as 3-phosphoglycerate, pyruvate, oxaloacetate or 2-oxoglutarate that are more oxidised than glucose. Their formation produces NADH. Most amino acids are more reduced than the intermediates, but the reactions leading to them from the intermediates almost invariably produce NADP. Apart from the histidine pathway, which is a net NADPH producer, and the pathways to glutamine, glutamate, tyrosine and phenylalanine, which neither consume nor produce NADPH, biosyntheses of all the other 15 amino acids from glucose produce between 1 and 8 moles of NADP per mole of amino acid and simultaneously produce NADH (Neidhardt et al. [1990]). Other reactions are then required to oxidise the NADH and reduce the NADP in order to achieve metabolic balance. This becomes a major factor with production organisms such as Corynebacteria modified and/or selected to produce huge amounts of amino acids on a commercial scale. To dispose of excess NADH, amino acid fermentations are operated under aerobic conditions, and oxygen is consumed in large amounts. To ensure maximum product formation, it is essential continuously to supply adequate amounts of oxygen, typically in the form of oxygen-enriched air (Hirose [1986]). Oxygen deficiencies, e.g., in high cell density fermentations or in cases where oxygen supplementation is uneconomical, typically result in lower product yields and productivities, as part of the carbon source is converted to compounds such as succinate, lactate or both to get rid of excess NADH.
Other examples include the enhanced biosynthesis of nucleotides, lipids and secondary metabolites by modified microorganisms selected or engineered to produce these compounds on the industrial scale. During these processes the microorganisms generally produce NADH and a central metabolic intermediate (such as pyruvate) that is more oxidised than the carbon source and reduce this intermediate to the desired product using NADPH. Once again, the microorganisms need to oxidise the excess NADH and reduce the excess NADP, and the yields on carbon source are decreased by the additional metabolic transformations of the carbon source required to achieve redox balance.
In all these examples, excess NADH is reoxidised either by respiration, requiring efficient aeration, or by the formation of unwanted side products, such as glycerol. Aeration on large industrial scales is expensive, and difficult to control exactly. In some processes, such as the fermentation of xylose to ethanol, reduction of excess NADP causes also problems. Important biochemical reactions regenerating NADPH are the oxidative branch of the pentose phosphate pathway (PPP), i.e. the successive reactions of glucose-6-phosphate dehydrogenase and 6 phosphogluconate dehydrogenase, and NADP-linked isocitrate dehydrogenase. Both reactions produce CO2. In industrial scale operations, this represents both a direct loss of carbon source and an environmental pollution. Furthermore, CO2 also acidifies culture media, necessitating the use of larger amounts of neutralising agents to control fermentation pH, and has a significant impact on cell physiology in general and amino acid production in particular. For example, CO2 inhibits enzymes in methionine and purine biosynthesis and has been reported to inhibit product formation in several fermentation processes including production of isoleucine, inosine, fumarate, penicillin and other antibiotics and yeast biomass (Hirose [1986]).
A general method to alleviate these problems without using aeration, which is expensive and difficult to control at optimal levels, would be very beneficial. Potential benefits include increased yields on carbon source, decreased energy consumption, significant decreases in CO2 production and increased specific productivity, which is particularly important in processes using immobilised microorganisms.
In the major routes of carbon and nitrogen metabolism it is a general rule that most catabolic pathways use the NAD/NADH coenzyme couple in the oxidation-reduction steps, whereas anabolic, synthetic reactions more frequently use the NADP/NADPH couple. Although the redox potentials (E′o) of these two couples are both close to −0.32 (Kaplan [1960]), the ratios of the reduced and oxidised forms of the two couples are maintained at very different levels in living cells. For example, in aerobic S. cerevisiae, NADH/NAD=0.9 and NADPH/NADP=3.2 (Sáez and Lagunas [1976]).
Most pyridine nucleotide dehydrogenases have a marked, often nearly absolute, specificity for one or the other pyridine nucleotide. Some dehydrogenases with the same substrate occur as both NAD- and NADP-specific enzymes. Usually only one of the enzymes is present under certain conditions, or the enzymes are expressed in different cell compartments. Good examples are glutamate dehydrogenases which are subject to complicated control mechanisms usually resulting in only one of the enzymes being dominant under any growth condition (e.g. Courchesne and Magasanik [1988]; Coschi-gano et al. [1991]; Miller and Magasanik [1991]; ter Schure et al. [1995]; Dang et al. [1996]; Avendano et al. [1997]). S. cerevisiae contains NAD- and NADP-linked isocitrate dehydrogenases: the cytosol (which is thought of as a single compartment) contains only NADP-linked enzyme and there is another NADP-linked enzyme in the peroxisomes whereas mitochondria (where the matrix, cristae and intermembrane space form three sub-compartments) contains both NAD- and NADP-linked enzymes (Minard et al. [1998]). Cells are therefore able to maintain NADH/NAD ratios much lower than the NADPH/NADP ratios, because reactions that transfer reducing equivalents between the two systems (and so would tend to equilibrate them) are restricted. Some bacterial and animal cells contain NAD(P) transhydrogenases (EC 1.6.1.1. and 1.6.1.2). Transhydrogenases are often membrane-bound enzymes with several subunits which are linked to energy production rather than to equilibration of the pyridine nucleotide systems. For the purposes of this patent application, the term “dehydrogenases” does not include the transhydrogenases EC 1.6.1.1 and 1.6.1.2. Many production organisms used in biotechnology, such as S. cerevisiae and Corynebacteria do not contain NAD(P) transhydrogenases, and so they appear to be unable to convert NADH plus NADP directly into NAD plus NADPH and vice versa.
The existence of two pyridine nucleotide systems and the absence of unregulated processes that would equilibrate them, suggests that the efficient growth and reproduction of presently evolved living organisms requires two distinct systems. The reason may be that a high NADPH/NADP ratio is required to drive biosynthetic reactions, whereas a lower NADH/NAD ratio is better suited for the generation of energy by pathways such as glycolysis and the tricarboxylic acid cycle (Metzler [1977]).
Boles et al. (1993) studied a mutant S. cerevisiae that lacked phosphoglucoisomerase, the enzyme that interconverts glucose-6-phosphate (Glc6P) and fructose-6-phosphate (Fru6P). This strain (a pgil—deletion mutant) is unable to grow on any hexose or pentose, though it can grow on certain mixtures of fructose and glucose (e.g. 2% fructose plus 0.1% glucose). The authors found that transformation of the mutant with a genomic library prepared from the mutant itself resulted in certain transformants that were able to grow on glucose alone, although 3- to 4-times slower than wild type, and contained plasmids comprising the GDH2 gene. This gene encodes an NAD-linked glutamate dehydrogenase. The authors argued that the simultaneous presence of substantial activities of both NADP- and NAD-linked glutamate dehydrogenases enabled the pgil—deletion mutant to grow on glucose by metabolising it through the PPP and converting the resulting NADPH into NADH, which could then be re-oxidised by functional mitochondria. Thus, these mutants were proposed to convert NAD plus NADPH into NADH plus NADP, which is the opposite transformation to that required of industrial production microorganisms (see above). Furthermore, their ability to survive on glucose was strictly dependent on the presence of functional mitochondria and oxygen and they were unable to ferment sugars into ethanol (Boles et al. [1993]).